Raman Tag

ABSTRACT

The present invention provides novel Raman tags for exploring membrane interactions in cells. The tags comprise a phenyl diyne probe where in the dyine is capped with the phenyl group. Methods for using the tags are also provided.

This invention was made with government support under CA182608 awardedby the National Institutes of Health. The government has certain rightsin the invention.

TECHNICAL FIELD

The present disclosure generally relates to tags for imaging moleculesusing Raman spectroscopy, and in particular to a method and compositionthat uses cholesterol mimics to track the location and movement ofcholesterol.

BACKGROUND

This section introduces aspects that may help facilitate a betterunderstanding of the disclosure. Accordingly, these statements are to beread in this light and are not to be understood as admissions about whatis or is not prior art.

An important component of cellular membrane, cholesterol controlsphysical properties of the membrane and contributes to specific membranestructures such as lipid rafts. Inside cells, cholesterol plays animportant role in various signaling pathways and serves as the precursorfor signaling molecules, and modifies specific proteins, such ashedgehog, to control protein trafficking and activity. The distributionof cholesterol in a living cell is highly regulated. Intracellularcholesterol is stored in lipid droplets (LDs) in the form of cholesterylester to avoid the toxicity caused by free cholesterol. Dysregulation ofcholesterol metabolism and/or trafficking has been linked to variousdiseases, including atherosclerosis, Niemann-Pick type C (NP-C) disease,and various cancers.

Intracellular cholesterol transport and metabolism have been studiedextensively using various reporter molecules, including cholesterolbinding molecules and cholesterol analogs. Cholesterol bindingmolecules, such as cholesterol oxidase, filipin, and perfringolysin Oderivatives, are commonly used to study steady-state distribution ofcholesterol in fixed cells and tissues. Fluorescent cholesterol,including intrinsic fluorescent sterols (DHE for dehydroergosterol) andfluorophore-tagged analogs (NBD-cholesterol and BODIPY-cholesterol) arewidely used in vitro and in vivo. Radiolabeled cholesterol or itsprecursors are used in biochemical studies of metabolism and traffickingof cholesterol.

These current cholesterol assays have limitations. Cholesterol oxidaseis commonly used in fluorometric or colorimetric assays to quantifytotal cholesterol in homogenized cells. Radiolabeled cholesterol has tobe used in combination with separation methods to determineintracellular cholesterol distribution indirectly. For imaging purpose,filipin is the most commonly used molecule for visualizing distributionof free cholesterol, but it is only applicable to fixed cells or tissueswith moderate specificity because filipin also labels other lipids.Fluorescent BODIPY-cholesterol is known to cause perturbations due tobulkiness of the fluorophore. DHE has the closest structure ascholesterol, but its fluorescence undergoes rapid photo-bleaching, whichimpedes real-time observation of cholesterol trafficking There exists aneed for new technologies that allow for real time imaging ofcholesterol transport and low toxicity in live cells.

SUMMARY OF THE INVENTION

The present invention provides novel Raman tags for exploring membraneinteractions in cells. The tags comprise a phenyl diyne probe where inthe dyine is capped with the phenyl group. Methods for using the tagsare also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a synthesis scheme and some resulting cholesterol mimics.

FIG. 2 depicts a spectral analysis of tagged cholesterol and SRSdetection of PhDY-Chol.

FIG. 3 is a graph illustrating the results of a cell-viability assayusing various concentrations of some of the generated cholesterolmimics.

FIG. 4 shows the SRS images of PhDY-Chol in live cells and what happenswhen PhDY-Chol storage in lipid droplets (LD) is blocked by an ACAT-1inhibitor, a schematic of how ACAT-1 modifies the cholesterol mimics,and a graph showing a quantitative analysis of PhDY-rich and BODIPY-richLDs in CHO cells before and after ACAT-1 inhibition.

FIG. 5 shows the cholesterol mimics transport and storage restored in anM12 cells disease model using HPβCD, a graph showing quanititativeanalysis of PhDY-rich areas in the cells before and after treatment withHPβCD, and a graph showing quantitative analysis of PhDY-Chol storage inLDs in cells before and after treatment with HPβCD.

FIG. 6 shows SRS images of PhDY-Chol visualized in various compartmentsof cholesterol storage in live C. elegans, in the presence and absenceof Cholesterol Uptake Protein-1 (ChUP-1).

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the principles of thepresent disclosure, reference will now be made to the embodimentsillustrated in the drawings, and specific language will be used todescribe the same. It will nevertheless be understood that no limitationof the scope of this disclosure is thereby intended. The terms “I,”“we,” “our” and the like throughout the Detailed Description,Appendix-A, and Appendix-B do not refer to any specific individual orgroup of individuals.

A novel composition and a method that allows bio-orthogonal imaging ofcholesterol esterification, storage, and trafficking inside living cellsand vital organisms. By rational design and chemical synthesis, weprepared a probe molecule, phenyl-diyne cholesterol (PhDY-Chol), whichgives a 2,254 cm⁻¹ Raman peak that is 122 times stronger than theendogenous C═O stretching band. Compared to alkyne-cholesterol mimic ofwhich the IC₅₀ is 16 μM, the phenyl-diyne group is biologically inertand did not cause cytotoxicity after 16 h incubation at 50 μM. In liveChinese hamster ovary (CHO) cells, SRS imaging showed incorporation intoplasma membrane, esterification of PhDY-Chol by acyl-CoA: cholesterolacyltransferase 1 (ACAT-1), and storage in lipid droplets (LDs). In acellular model of NP-C disease, PhDY-Chol is selectively accumulated inlysosomes and is esterified and relocated to LDs after treatment with acholesterol-mobilization drug. In live C.elegans, SRS imaging ofPhDY-Chol revealed a previously unnoticed compartment of cholesterolstorage, regulated by the cholesterol uptake protein ChUP-1. Thesestudies herald the potential of the method for unveiling intracellularcholesterol trafficking mechanisms and highly efficient screening ofdrugs that target cholesterol metabolism.

The following describes compositions, methods of synthesis, and methodsof use of these compositions in the study of cholesterol. Thesecompositions may be used to study cholesterol localization and movementwithin live cells. The cholesterol mimics may be used to study andunderstand different changes in metabolism and organization by trackingthe changes in localization and modification of cholesterol. Thecholesterol mimics may be used to generate assays for screening leadcompounds or treatments to prevent or treat a metabolic irregularity ordisease such as cancer. In other aspects the cholesterol mimic may beused to study the lipid droplets with in a given cell, healthy or nothealthy, to provide an analysis. The cholesterol mimcs may be used as atarget in a drug delivery system, where the drug targets the mimic in acertain unmodified or modified state, specifically. The cholesterolmimics may be used to provide a diagnosis or monitor the health of apatient's cells before or during treatment for a disease. Thecholesterol mimics may be used in vitro or in vivo (i.e. cells andtissues, whole organs, or live animals or humans), and may be analyzedin live or fixed cells.

The cholesterol mimics may be provided for use in a powder or crystalform, or suspended in liquid. The cholesterol mimics may be supplied byseveral delivery means including but not limited to orally,intravenously, injection, inhalation, catheter, dermal absorption, oringestion.

In general, a cholesterol mimic is generated by replacing the aliphaticchain of in cholesterol with a group that changes the Raman spectraproduced during Raman spectroscopy. Ideally, a peak is generated fromthis molecule that is differentiated from other signals produced by acell or tissue. One aspect, is to replace the aliphatic chain ofcholesterol with a group that will generate a peak between 1,800 and2,800 cm⁻¹ in a Raman scatter. One way to produce a signal in thisregion is to add a C≡C moiety to cholesterol, as shown in FIG. 1. Incertain aspects a C≡C bond may be added one, two, three, four, five, orsix times to increase signal. In certain aspects there are three C≡Cbonds included in the cholesterol mimic.

In certain aspects the PhDY-Chol mimic probe was generated by replacingthe aliphatic chain in cholesterol with phenyl-diyne. The followingchemical structures illustrate some of the mimics:

In certain aspects of the technology, the mimic is viewed by Ramanspectroscopy. Those of skill in the art will understand the set up andlaser sources to use given the chemical nature of the cholesterol mimic.For illustrative purposes only, one may look to the contents ofAppendix-A, herein incorporated by reference in its entirety, tounderstand one example of a setup for performing Raman spectroscopyusing the disclosed cholesterol mimics.

Cholesterol mimics were synthesized and a few specific embodiments willnow be described. Referring now to FIG. 1, alkyne cholesterol (A-Chol,5), phenyl-alkyne cholesterol (PhA-Chol, 6), phenyl-diyne cholesterol(PhDY-Chol, 7), and cyano cholesterol (CN-Chol, 8). Initially, synthesiscommenced with cholenic acid 3. Using a sequence of THP-protection,LiA1H₄ reduction, Dess-Martin oxidation and Seyferth-Gilbert-Bestmannhomologation, cholenic acid 3 was converted to compound 4 intermediatewith a terminal alkyne group. Removal of the THP-protecting group gaveprobe A-Chol 5. PhA-Chol 6 and PhDY-Chol 7 were prepared from compound 4intermediate via a palladium-catalyzed Sonogashira reaction and acopper-catalyzed Cadiot-Chodkiewicz reaction, respectively, followed byacidic removal of THP group. Additionally, CN-Choi 8 was prepared fromcholenic acid 3 via standard transformations.

The following are specific examples and embodiments, and not meant to belimiting in any way.

Design and Synthesis of Composition

Referring to FIG. 1, in order to design a probe molecule that not onlymaintains physiological functions of cholesterol, but also has a largeRaman scattering cross section, we chose to replace the aliphatic sidechain of cholesterol with a cyano or alkynyl groups. These groups havesmall size, which could minimize structural perturbation of the moleculeof interest, in this case, cholesterol. These groups produce strongRaman scattering peaks in a cellular silent region (1,800-2,800 cm⁻¹),and can be used for Raman imaging in a low-concentration condition. Ithas been reported that as the chain length increases, thehyperpolarizability increases in polyynes. Also, aromatic ring cappedalkyne was shown to give stronger Raman signals than terminal alkyne. Todesign cholesterol mimic with very strong Raman intensity, we calculatedthe Raman cross section of potential tags—alkyne, phenyl-alkyne, diyne,phenyl-diyne using the Q-Chem and GAMES S electronic structure packagesto provide insight of the relation between molecular structure and Ramanintensity. The results showed that the localized polarizabilities oneach C≡C moiety increase with the number of conjugated triple bonds, aswell as with addition of a phenyl ring.

The total polarizability of the molecule increases as a result of theadditive effect as well as non-linear boost in the polarizability ofconjugated bonds. The phenyl ring serves as both a donor and an acceptorof π-electrons from the neighboring triple bonds, further escalatingpolarizabilities of neighboring conjugated bonds. Taking into accountthat the Raman intensity is proportional to squares of polarizabilityderivatives, the additional three-fold enhancement of the totalpolarizability due to conjugation results in a ˜10-fold boost in Ramanintensity. Together, the Raman intensity increases further by adding aphenyl group to the terminal alkyne, and increases even further byconjugating a phenyl group and another alkyne.

Raman Spectral Analysis of Stimulate Raman Scattering of CholesterolMimics

Referring now to FIG. 2, to determine the Raman shift of the C≡Cstretching vibrational mode and to compare the level of Raman signalsfrom the cholesterol mimics, 50 mM of each compound was prepared incyclohexanone and confocal Raman spectral analysis was performed. Thesignal from CN-Chol was too weak to be detected. A-Chol showed its peakfor C≡C vibrational mode at 2,122 cm⁻¹; PhA-Chol at 2,239 cm⁻¹;PhDY-Chol at 2,254 cm⁻¹. Comparing the Raman peak of each tag to the1,714 cm⁻¹ C═O vibrational peak from the solvent (9.7 M for purecyclohexanone), the alkyne, PhA, and PhDY groups were 11 times, 16times, and 122 times stronger in Raman intensity, respectively. Thisshowed that the PhDY tag produces a spectrally-isolated peak, which isstronger than the C═O vibrational mode by two orders of magnitude.

To determine the SRS imaging sensitivity for PhDY-Chol, we used afemtosecond stimulated Raman loss (SRL) microscope reported elsewhere.Cyclohexanone solutions of PhDY-Chol were prepared by serial dilution,and SRS images of PhDY-Chol were recorded with the laser beatingfrequency tuned to be resonant with C≡C vibration at 2,252 cm⁻¹. Insolutions without PhDY-Chol, a residual background was detected, causedby cross phase modulation. The SRS contrast, defined as (S—B)/B, where Sand B denote SRS signal and background, was calculated as a function ofPhDY-Chol molar concentration. At the speed of 200 μs as per pixel, alinear relationship was observed and 13% and 4% contrasts were reachedat 313 μM and 156 μM, respectively. To increase the detectionsensitivity, we chirped the femtosecond lasers to 0.8 picosecond with aSF-10 glass rod. This spectral focusing approach maintained 85% of theSRS signal while reduced the cross phase modulation background level by3 times, to a level of 6.3×10⁻⁷ in terms of modulation depth. As aresult, the SRS contrast became 14% at 31 μM, corresponding to ˜1,800molecules in the excitation volume. We also depicted the modulationdepth (ΔI/I) as a function of molar concentration (Appendix-B), which isused for estimating the molar concentration of PhDY-Chol inside cells infollowing studies.

Cytoxicity Analysis of the Cholesterol Mimics

Referring now to FIG. 3, the cytotoxicity of cholesterol mimics wasevaluated by MTT cell-viability assays after treating CHO cells withcholesterol mimic. Various concentrations of cholesterol mimic wereadded to the culture media and the cells were incubated for 48 hoursbefore the assays were conducted. A-Chol was found to be toxic to thecells with IC₅₀ of 16 μM. Adding a phenyl group reduced thecytotoxicity. To directly visualize the toxic effect, we stained thecells with propidium iodide for late apoptosis and necrosis. Cellsincubated with A-Chol showed reduced density and extensive apoptosis,whereas both PhA and PhDY caused minimum cell death (Appendix-B). Thisresult presents another important role of the phenyl group, which is toreduce the toxicity caused by terminal alkyne.

Membrane Incorporation of Cholesterol Mimics

Referring now to FIG. 4, CHO cells which are commonly used forcholesterol trafficking and metabolism studies, were used in thesestudies. To enhance cellular uptake of PhDY-Chol, the cells werepre-incubated in medium supplemented with lipoprotein-deficient serum todeplete medium cholesterol, after which the cells were incubated with 50μM PhDY-Chol for 16 hours. By tuning the laser beating frequency to beresonant with C≡C vibration (2,252 cm⁻¹), SRL signals arose fromPhDY-Chol. We also tuned the laser to be resonant with C—H vibration(2,885 cm⁻¹) and obtained signals from C—H-rich lipid structures, suchas LDs.

To show the incorporation of PhDY-Chol into the plasma membrane, thecells were trypsinized and performed spectral-focusing SRS imaging ofthe rounded live CHO cells with 10 μs per pixel speed. PhDY-Chol in themembrane was detected in the on-resonance image, and the contrastdisappeared in the off-resonance image. The membrane incorporation wasconfirmed by filipin staining of free cholesterol and Raman spectralanalysis (Appendix-B). By focusing at the filipin-stained membrane, wehave obtained the Raman spectrum showing the C═C band from filipin, theamide I band from protein, and the C≡C band from the PhDY (Appendix-B).Inside live CHO cells, PhDY-Chol was colocalized with LDs found in theC—H vibrational region, as shown in FIG. 4. This colocalization wasconfirmed by two-photon-excited fluorescence (TPEF) imaging and Ramanspectral analysis of BODIPY-stained LDs in fixed CHO cells.(Appendix-B). The Raman spectrum of the BODIPY-labeled LDs showed theC═C band from BODIPY, 702 cm⁻¹ peak from cholesterol ring, and the C≡Cband from the PhDY (Appendix-B), which further supports the localizationof PhDY-Chol in LDs.

It is important to note that PhDY-Chol-rich structures inside the CHOcells could not be stained by filipin (Appendix-B), indicating that itis not in the free form. It is hypothesized that PhDY-Chol is convertedinto PhDY-cholesteryl ester, by ACAT-1, the enzyme responsible forcholesterol esterification, as diagrammed in FIG. 4. To confirm theesterification of PhDY-Chol, we inhibited ACAT-1 with avasimibe for 24 hbefore addition of PhDY-Chol. After blocking cholesterol esterification,the amount of PhDY-Chol found in CHO cells significantly decreased (FIG.4 c). Although LDs were still visible, the amount of PhDY-Chol signalfound inside LDs reduced by 4 times (FIG. 4 d). ACAT-1 knockdown byshRNA was also conducted to specifically inhibit the enzyme. Similarly,we found decreased amount of PhDY-Chol in ACAT-1 knocked down CHO cells,and the amount of PhDY-Chol in LDs reduced significantly (Appendix-B).To determine where PhDY-Chol accumulates after ACAT-1 inhibition, westained the cells with LysoTracker for lysosomes. Our results indicatedthat after ACAT-1 inhibition, PhDY-Chol partially located in lysosomes(Appendix-B). Collectively, these results show that PhDY-Chol can betransported into cells, converted into PhDY-cholesteryl ester by ACAT-1,and stored in LDs following the normal metabolic pathway of cholesterol.To emphasize the physiological compatibility of our PhDY tag, we treatedCHO cells with BODIPY-cholesterol. The amount of BODIPY-cholesterolincorporated into LDs did not change after ACAT-1 inhibition (FIG. 4 dand e), indicating that BODIPY-cholesterol directly labels the LDswithout metabolic conversion into cholesteryl ester.

Lysosomal accumulation and relocation to Lipid Droplets in NP-C animaldisease model discovery using cholesterol mimics.

Referring now to FIG. 5, the potential of PhDY-Chol for studyingcholesterol transport in NP-C disease, a disorder featured by abnormalcholesterol accumulation in late endosome/lysosome caused by mutation inNPC1 or 2 gene, was explored. M12 cells, mutant CHO cells that contain adeletion of the NPC1 locus, were established as a cellular model of theNP-C disease. By combining SRL imaging of PhDY with TPEF imaging offilipin, we observed that, unlike wildtype CHO cells, the PhDY-Chol-richstructures were stained by filipin, indicating that these PhDY-Cholmolecules were located in lysosomes. (Appendix-B). Moreover, we observedsome filipin labeled structures that do not contain PhDY-Chol. Thisresult is reasonable given that filipin has been shown to label otherlipid molecules, such as glycosphingolipids. As additional evidence, weincubated M12 cells with PhDY-Chol and stained the cells withLysoTracker. It was found that all PhDY-Chol-rich areas were localizedin LysoTracker-stained organelles (Appendix-B). Collectively, theseresults showed that PhDY-Chol can selectively represent the lysosomalstorage of cholesterol in the NP-C disease model.

We then treated the PhDY-Chol-labeled M12 cells with acholesterol-mobilizing drug, hydroxypropyl-β-cyclodextrin (HPβCD). Thisdrug is known to mediate lysosomal escape of cholesterol, and promotestorage of excess cholesterol into LDs. After treating with HPβCD, theamount of PhDY-Chol in M12 cells decreased by half (FIG. 5 b and c).Interestingly, we observed that some PhDY-Chol-rich areas were notlabeled by filipin after HPβCD treatment (FIG. 5 b, arrow heads). Theseareas likely represent PhDY-cholesteryl ester stored in LDs. To confirmthis possibility, we stained the cells with BODIPY for localization ofLDs. The result clearly showed that PhDY-Chol moved into LDs after HPβCDtreatment, and the number of PhDY-rich LDs increased significantly(FIGS. 5 d and e). Together, these data indicate that PhDY-Chol can beused as a reliable probe molecule to study cholesterol mobilizationinside living cells.

Cholesterol mimics identify cholesterol storage compartments in animalmodel.

Referring to FIG. 6, to demonstrate the capability of monitoringcholesterol uptake and distribution in vivo, we fed N2 wildtype C.elegans with PhDY-Chol-labeled E. coli and imaged PhDY-Chol storage inthe worms using our SRL microscope at speed of 40 μs per pixel.PhDY-Chol was found to be stored in the intestinal cells inside thewildtype worms (FIG. 6 a, upper panels). To confirm the uptake ofPhDY-Chol by intestinal cells, we fed mutant C. elegans, in whichdietary cholesterol uptake is inhibited by ChUP-1 deletion, withPhDY-Chol. We did not observe PhDY-Chol inside this strain (FIG. 6 a,lower panels), which indicates that the PhDY tag did not affect thecholesterol uptake process. Then, we tuned the laser to be resonant withC—H vibration for lipid-rich LDs. Unlike wildtype CHO cells, thePhDY-Chol-rich compartments were found to be distinguished from LDs inwildtype worms (FIG. 6 a, upper panels). To explore the nature of thesecompartments, we used hjIs9 worms that contains GFP targeted tolysosome-related organelles (LROs) in intestinal cells. Dual-modalitySRS and TPEF imaging showed that PhDY-Chol is stored in the LROs (FIG. 6b). Collectively, these results suggest that dietary PhDY-Chol uptake isthrough a ChUP-1 mediated process, and unlike mammalian CHO cells, C.elegans stores cholesterol in LROs, but not in LDs in the intestine.

Chemical structure (V) or Compound S8. The mixture of 4 (22 mg, 0.05mmol), PdCl ₂ (PPh₃)₂ (1.4 mg, 0.002 mmol), CuI (0.4 mg, 0.002 mmol),and B (12.3 mg, 0.06 mmol) in THF (0.5 mL) was bubbled with Argon gasfor ten minutes. To the mixture, DIPEA (0.02 mL) was added at roomtemperature. After stirring for 2 h at room temperature, the reactionwas quenched with saturated NH₄Cl aqueous solution and extracted withethyl acetate. The organic layer was washed with brine, dried overMgSO₄. The solvent was removed under vacuum, and the residue waspurified by chromatography (Hexane/EtOAc, 40:1) to give S8 (18 mg, 65%)as a white solid.

¹H NMR (500 MHz, CDCl₃): δ 7.50 (d, J=7.0 Hz, 2H), 7.38-7.35 (m, 1H),7.33-7.30 (m, 2H), 5.35 (t, J=6.5 Hz, 1H), 4.72 (m, 1H), 3.93-3.90 (m,1H), 3.55-3.47 (m, 2H), 2.44-2.20 (m, 4H), 2.01-1.96 (m, 2H), 1.88-1.83(m, 4H), 1.74-1.70 (m, 2H), 1.62-1.42 (m, 12H), 1.30-1.06 (m, 7H), 1.01(s, 3H), 0.93 (d, J=6.5 Hz, 3H), 0.69 (s, 3H); ¹³C NMR (100 MHz, CDCl₃):δ 141.2, 141.1, 133.1, 129.6, 128.6, 121.7, 121.6, 121.4, 108.8, 97.2,97.0, 83.3, 76.2, 75.5, 74.8, 67.6, 65.6, 63.1, 63.0, 59.6, 56.9, 56.0,53.6, 50.3, 50.3, 42.6, 40.4, 40.0, 38.9, 37.6, 37.4, 37.0, 36.9, 35.4,34.5, 32.0, 31.4, 29.8, 28.3, 28.1, 25.6, 24.4, 21.2, 20.3, 20.2, 19.5,18.3, 16.8, 12.0; IR (film): 2958, 2925, 2326, 2125, 1643, 1457, 1379,1016 cm⁻¹; MS (ESI): m/z 585 [M+Na]⁺.

Additional disclosure is found in Appendix-A, Appendix-B, and Appendix-Cfiled herewith, entirety of each of which is incorporated herein byreference into the present disclosure.

Those skilled in the art will recognize that numerous modifications canbe made to the specific implementations described above. Theimplementations should not be limited to the particular limitationsdescribed. Other implementations may be possible.

While the inventions have been illustrated and described in detail inthe drawings and foregoing description, the same is to be considered asillustrative and not restrictive in character, it being understood thatonly certain embodiments have been shown and described and that allchanges and modifications that come within the spirit of the inventionare desired to be protected.

What is claimed is:
 1. A composition of the following formula:


2. A method of use as shown and described.